1. Field of the Invention
The present invention relates to electrodes for detecting uric acid and their producing methods.
2. Description of Related Art
Commonly found in urine and blood, uric acid (UA) is the major end product of purine metabolism in human body. In healthy people, the uric acid level in serum is in the range of 0.24-0.52 mM and is in the range of 0.214-4.40 mM in urine. High uric acid concentration accumulated in serum or urine that exceeds the normal level leads hyperuricemia and induces gout, Lesch-Nyhan syndrome, and renal diseases. Therefore, it is important to monitor uric acid in serum or urine regularly for precaution, diagnosis, and treatment of those diseases caused by high concentration of uric acid.
The analytical methods for uric acid include spectrophotometry, enzymatic method, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis, chemiluminescence, fluorescence, phosphorescence, and electrochemical methods. But these methods generally involve tedious separation procedure, need expensive instrument, and lack specificity. Therefore, there is a great interest in developing a method that is specific, simple, easy operation, fast, and low cost for uric acid analysis. The enzyme-based sensor electrode biosensor which allows direct, specific, rapid, sensitive, and inexpensive measurement of uric acid fulfills these criteria. The use of uricase (urate oxidase, UOx) for detecting uric acid can be traced back to the work of John and Bulger in 1941 [1].
To set up a stable long-term reusable uricase electrode, the immobilization of uricase on the electrode is the most important factor. There are many enzyme immobilization methods such as the physical adsorption method [2-7], the entrapment method [8-9], the self-assembled monolayer (SAM) method [10-11], the cross-linking method [12-13], and the covalent chemical bonding method [14-15]. Wherein the simplest physical adsorption subjects the problem of easily peeled off from the electrode that causes poor electrode life. There is similar problem with the entrapment method. Although the self-assembly monolayer method exhibits many merits, the long-term application is still limited. Therefore, the cross-linking method or the covalent chemical bonding method could be the ideal method for uricase immobilization to enhance the electrode performance and shelf life.
The electrode material also plays very important role for the enzyme sensor electrode which closely related to the enzyme immobilization, current conduction, and electrode fabrication costs. Metals and non-metal carbon are the two major categories. Modified metal electrodes such as zinc oxide coated gold electrode [6], the multi-walled carbon nanotube (MWCNT) modified gold electrode [15], carboxylated MWCNT and polyaniline (PANI) conducting polymer modified gold electrode [5], CuO film modified platinum electrode [3], and gold nanoparticles (AuNPs) over SAM 3-aminopropyltriethoxysilane (APTES) coated indium tin oxide (ITO) electrode [14] have been extensively used nowadays for uricase electrode. Since metal electrodes are generally expensive, carbon-based non-metallic electrodes has been gradually more interested by researchers for fabricating uricase electrode, for examples, self-assembled UOx mixed thionine-graphene oxide hybrid nanosheets on glassy carbon electrode [11], graphite disc electrode modified with Prussian blue-Ni2+ film [16], sandwich format amperometric screen-printed carbon electrode [4], ferrocene modified grapheme oxide screen printed electrode (SPE) [7], and screen printed Ir-modified carbon electrode [13].
Uric acid is an electroactive compound that can be easily oxidized at the electrode by UOx in aqueous solution to produce allantoin, H2O2, and CO2 [17]:

The produced H2O2 can be detected by its oxidation at the surface of working electrode.H2O2→O2+2H++e−
However, the required electrochemical oxidation potential for H2O2 is usually high (>0.40 V) which leads other electroactive species such as ascorbic acid also oxidized in the solution to interfere the uric acid analysis. One of the approaches to solve this problem is the use of redox mediator for substituting O2 to decrease the oxidation potential of uric acid to avoid the unnecessary interferences. In the meantime, the variation of atmospheric pressure during the electrochemical measurements which affects the accuracy of analysis can also be avoided. Recently, ferrocene and its derivatives have been used as a redox mediator in many applications. For instances, electropolymerization of pyrrole on Pt electrode surface containing ferrocene as a redox mediator was carried out to form a polymer film which was used to immobilized UOx for the determination of uric acid in biological fluids [17]; ferrocene carboxaldehyde was chemical bound to graphene oxide and coated over SPE to immobilize UOx for the determination of uric acid in serum [7].
In addition, the previous application (application Ser. No. 12/421,565) of the inventors provided an enzyme electrode having good performance.